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ORIGINAL ARTICLE
M. Mohri Æ H. A. Seifi Æ S. H. Zamani Sani
Effects of oral administration of levamisole on non-specific immunity,
serum proteins and health in normal colostrum-fed neonatal dairy calves
Received: 12 September 2004 / Accepted: 2 November 2004 / Published online: 14 December 2004
Springer-Verlag London Limited 2004
Abstract Soon after levamisole became widely used as a
veterinary anthelmintic, it was recognised that concur-
rent enhancement of immune responsiveness sometimes
accompanied anthelmintic treatment, especially in old or
chronically ill animals. There are many reports con-
cerning the immunomodulatory effects of levamisole in
immunocompromised and vaccinated animals, but
information about the effects of levamisole in immuno-
competent animals is limited and controversial. Thirty
Holstein dairy calves were randomly divided into two
groups: test (n=15) and control (n=1 5). Blood samples
were taken from the jugular vein, between 24 h and 48 h
after birth, and put into EDTA-containing tubes and
plain tubes; levamisole was then administrated orally at
a dose of 2 mg/kg body weight three times at 1-day
intervals in test groups. Blood sampling from all the
calves was continued at days 7, 14, 21 and 28. The
weight of calves was measured only at days 1 and 28
(before feeding). The levels of packed cell volume (PCV)
(microhaematocrit), total white blood cells (WBCS) and
differential leukocyte count (manual standard methods),
total serum protein (colorimetry), beta and gamma
globulins (electrophoresis), albumin:globulin (A:G)
(calculated) and disease occurrence were measured and
recorded. Appropriate statistical methods were used for
data analysis, and P £ 0.05 was considered as signifi-
cant. No significant differences were seen for PCV, total
serum protein, WBC count and health between groups.
In the test group, neutrophil level at day 14 and
monocyte level at day 21 were significantly higher than
in the control group (P<0.05). Electrophoresis showed
no significant differences for beta globulin level and A:G
between groups, but gamma globulin level at day 28 was
significantly higher in the test group (P<0.05).
Keywords Dairy calves Æ Health Æ Leukogram Æ
Levamisole Æ Se rum proteins
Introduction
Levamisole is a highly acceptable anti-nematodal drug
because of its broad range of activity in a large number
of hosts (sheep, cattle, horse, pig, dog, chicken). Soon
after tetramisole and, subsequently, levamisole became
widely used as veterinary anthelmintics, it was recognised
that concurrent enha ncement of immune responsiveness
sometimes accompanied anthelmintic treatment, espe-
cially in old or chronically ill animals (Courtney and
Roberson 1995).
The high mortality of infant cattle, horse, sheep and
pigs has been a serious problem in farm animal breeding
for many years. Such losses are mostly caused by
infectious diseases, especially in the neonatal and post-
natal periods.
Intensive studies have recently been initiated to assess
methods of preventing and treating infectious diseases.
One of these methods has been non-specific immuno-
prophylaxis with natural or synthetic immunomodula-
tors. Successful results have already been obtained in the
prophylaxis of diseases of young farm animals after non-
specific stimulation of the pregnant adults (Deshpande
et al. 1991; Kotowski 1991; Krakowski et al. 1999;
Wawron and Szczubial 2000).
These immunologically enhancing treatments sug-
gested that the drug might be of value in promoting cell-
mediated and humoral immunity in neonatal dairy
calves. Most previous reports studied the effects of lev-
amisole administration on immunocompromised or
vaccinated animals (Irwin et al. 1976; Babiuk and Misra
1981, 1982; Roth and Kaeberle 1984; Ivanov et al. 1987;
Ogunbiyi et al. 1988; Sharma et al. 1990), but there are
M. Mohri (&) Æ H. A. Seifi Æ S. H. Zamani Sani
Department of Clinical Sciences,
School of Veterinary Medicine,
Ferdowsi University of Mashhad,
91775-1793, Mashhad, Iran
E-mail: mohri@ferdowsi.um.ac.ir
Tel.: +98-511-6610430
Fax: +98-511-6620166
Comp Clin Path (2005) 13: 132–136
DOI 10.1007/s00580-004-0528-0
limited data concerning the effects of orally administered
levamisole in normal immunocompetent neonatal
calves. The present study was performed to reveal the
effects of levamisole on leukogram, globulin levels and
health in normal colostrum-fed neonatal dairy calves.
Materials and methods
The study was conducted in a da iry herd with approxi-
mately 400 calves per year at Mashhad suburb (north-
east of Iran). This herd consisted of pure-bred animals
of the Holstein breed. The herd was totally confined in
free-stall housing without access to pasture. Cows were
fed with alfalfa hay, concentrate and corn silage.
The cows were dried 2 months before their expected
time of parturition and transferred to a separate stall. As
the time of parturition approached, they were moved to
straw-bedded maternity pens. Prompt assistance was
given to cows with dystocia. Following parturition the
umbilicus of each calf was treated with pavidone iodine,
and the calf was weighed and transferred to an indi-
vidual pen. Within the first 6 h of life 2.5 kg of the dam’s
colostrum was bottle-fed to the calf, and colostrum
feeding was cont inued every 12 h for 48 h. After 48 h
herd milk was used for feeding twice daily (2 kg every
12 h) until the calf was 10 days old. After this time
concentrate, high quality alfalfa and water were allowed
ad libitum. The calves were weaned at 45 days of age.
The heifer calves were mainly used as herd replacements.
Thirty calves were used in the present study. They
were divided into two groups (test n=15; control n=15).
Based on the gender and parity of dams, the number of
calves in each group was approximately identical. In the
test group oral levamisole was administered at a dose of
2 mg/kg body weight three times at 1-day intervals.
Samples were taken from the jugular vein between 24 h
and 48 h after birth (before levamisole administration in
the test group) and on days 7, 14, 21 and 28. The blood
was added to EDTA-containing tubes for haematologi-
cal measurements and plain tubes for serum extraction
and measurement of total protein and serum protein
electrophoresis. The serum was harvested after centri-
fugation at 3,000 rpm for 10 min and stored at 20C
until required for analysis.
The haematocrit [packed cell volume (PCV)] levels
were obtained by microhaematocrit methods. White
blood cell (WBC) measurements were taken manually by
standard methods (Dacie and Lewis 1984). Differential
leukocyte counts were performed on routinely prepared
Giemsa-stained blood films by the cross-sectional tech-
nique (Jain 1986). One hundred leukocytes were identi-
fied. Serum total protein was determined by Biuret
colorimetric method with a spectrophotometer (Jenway
6105, Jenway, Felstead, England). Percentage and
absolute levels of beta and gamma globulins were
determined by cellulose acetate electrophoresis and
densitometry (Helena system). Disease occurrence was
also recorded during the experiment.
The SAS program was used for data analysis. After
testing normal distributions of the data we used a
parametric independent t-test to investigate significant
difference between groups at various sampling times.
The chi-square test was also used for comparison of
disease occurrence between groups. P £ 0.05 was con-
sidered as signific ant.
Results
The results are shown in Tables 1 and 2. No significant
differences were seen for PCV, total serum protein and
WBC count between groups. In the test group, neutro-
phil level at day 14 and monocyte level at day 21 were
significantly higher than the control group (P<0.05).
Electrophoresis showed no significant differences for
beta globulin level and albumin:globulin (A:G) between
groups but gamm a globulin level at day 28 was sig-
nificantly higher in the test group (P<0.05).
During the study, in the test group, six cases of
diarrhoea were recorded. In the control group ten cases
of diarrhoea occurred. The chi-square test showed no
significant difference between groups for disease occur-
rence.
Discussion
There are reports concerning the effects of levamisole
administration on the immune response of various ani-
mal species (Hunter et al. 1981; Singh and Dhawedkar
1993; Sopinska and Guz 1993; Wisniewski et al. 1993;
Asif et al. 1995; Cabaj et al. 1995; Keskar et al. 1996;
Findly and Munday 2000; Qureishi et al. 2000; Zia-ul-
Rahman et al. 2003). For calves, most of the papers
studied the immunomodulatory effects of simultaneous
administration of levamisole and various vaccines.
However, the information about the effects of levamisole
in normal intact neonatal dairy calves is limited and
controversial.
There are few reports concerning the alterations of
leukocyte number after levamisole administration. Asif
et al. (1995) reported no significant changes in the
leukocyte, neutrophil, eosinophil and basophil counts in
Sahiwal heifers after oral administration of levamisole.
In another study on buffalo heifers, Zia-ul-Rahman et al.
(2003) reported an increase of WBCs on day 1 after
levamisole administration, which returned to pre-dose
levels at 7 and 14 days after dosing. Neutrophil per-
centage decreased and lymphocyte percentage increased
on days 7 and 14 after drug administration, but an
increase in monocyte count was observed on days 7 and
14 of the experiment. Our results are consisten t with
those of Asif et al. (1995) and Zia-ul-Rahman et al.
(2003), with regard to WBC count, and Zia-ul-Rahman
et al. (2003) for monocyte level. Paulik et al. (1992)
showed increased levels of neutrophil percentage in
mice after levamisole administration. Nalini-Kumari and
133
Choudhuri (1986) have studied the levamisole effects,
together with rinderpest vaccinat ion, in buffalo calves.
They reported a non-significant increase in leukocyte
count, with significant increase in lymphocyte percentage
that was associated with a decrease in the neutrophil le-
vel. These changes occurred 1–3 weeks after levamisole
administration. Goranov and Bonovska (1987) reported
no changes in the total count of leukocytes in sheep after
levamisole injection, although a rise in the phagocytic
activity of neutrophils was seen. In the present study
there were significant differences between neutrophil and
monocyte levels at the second and third weeks of the trial
between groups, respectively (P<0.05). These findings
are consistent with those of Paulik et al. (1992) but are
in contrast with the results of Nalini-Kumari and
Choudhuri (1986) and Goranov and Bonovska (1987).
This study revealed significant difference in gamma
globulin levels between groups, with higher values in the
test group (P<0.05). Since most of the im-
munoglobulins migrate in the gamma globulin zone, this
suggests that levamisole administration induced anti-
body production in the test group.
Some apparently conflicting evidence also exists with
regard to the effect of levamisole on antibody produc-
tion. The results of the present study are consistent with
Bekere’s findings (1988), indicating that levamisole given
to neonatal cattle increases the value of humoral factors
of natural immunity, and levamisole could, therefore, be
used for improving the formation of natural immunity
and as a preventive against hypoproteinaemia in new-
born cattle. Zia-ul-Rahman et al. (2003 ) reporte d in-
creased levels of total serum protein on day 7, whereas
globulin increased on days 7 and 14 after levamisole
administration in buffalo heifers. Our results are con-
sistent with those of Zia-ul-Rahman et al. (2003) for
gamma globulin level. The difference between times of
increase is probably related to species difference or dose
and number of administrations. Singla and Juyal (1992)
observed an increase in serum gamma globulin levels in
calves infected with Trypanosoma evansi and in calves
infected with T. evansi and simultaneously treated with
levamisole, but, compared with untreated uninfected
controls, the increase was significant only in the infected/
treated group. Furthermore, there are reports concern-
ing augmented humoral immune responses after lev-
amisole administration following vaccination against
Pasteurella multocida and infectious bovine rhinotra-
cheitis virus in calves and cattle, respectively (Babiuk
and Misra 1982; Sharma et al. 1990). Similar results
were obtained in suckling rats immunised against sheep
red blood cells (Hunter et al. 1981). Sheep given vacci-
nations against clostridial diseases and levamisole had
higher anti-clostridial antibody titres than sheep given
only vaccine (Hogarth-Scott et al. 1980 ). On the other
hand, in a further study, blood derivative containing
10% bovine serum proteins (60% immunoglobulins
and 40% albumin) and 10 mg levamisole per ml, or
levamisole alone, was given i.m. to fifteen 3-week-old
calves at a levamisole dose of 10 mg/kg body weight, the
Table 1 Mean ± SE of some haematological parameters and statistical comparisons between trial groups
Timepoint PCV (%) WBC(10
9
/l) Neutrophils (10
9
/l) Band(10
9
/l) Lymphocytes (10
9
/l) Monocytes (10
9
/l) Eosinophils (10
9
/l)
Test Control Test Control Test Control Test Control Test Control Test Control Test Control
24–48 h 35.11±0.02 33.31±0.01 10.21±1.08 9.74±0.86 6.04±0.59 6.64±0.70 0.000 0.004±0.004 4.04±0.65 2.93±0.26 0.080±0.049 0.086±0.043 0.014±0.006 0.053±0.035
P value 0.76 0.87 0.52 0.4 0.31 0.78 0.38
First week 32.01±0.01 32.11±0.01 8.39±0.74 10.73±1.18 3.44±0.62 5.08±1.02 0.005±0.005 0.006±0.006 4.69±0.32 5.22±0.80 0.081±0.022 0.055±0.025 0.016±0.009 0.011±0.011
P value 0.62 0.28 0.32 0.1 0.74 0.28 0.37
Second week 30.41±0.01 31.00±0.01 8.66±0.98 8.22±0.68 3.74±0.59 2.36±0.50 0.000 0.006±0.006 4.84±0.48 5.53±0.48 0.12±0.044 0.070±0.029 0.000 0.000
P value 0.78 0.98 0.02 0.36 0.32 0.34 –
Third week 29.81±0.01 29.31±0.01 9.38±0.78 8.18±0.59 2.60±0.42 2.25±0.34 0.000 0.000 6.48±0.48 5.54±0.41 0.17±0.071 0.049±0.043 0.000 0.015±0.009
P value 0.84 0.23 0.07 – 0.12 0.01 0.09
Fourth week 29.31±0.01 31.11±0.02 10.09±0.91 9.68±0.74 3.63±0.49 3.05±0.43 0.000 0.000 6.39±0.61 6.50±0.41 0.077±0.035 0.10±0.029 0.000 0.000
P value 0.82 0.82 0.39 – 0.59 0.35 –
134
dose being repeated 5 days later. Neither preparation
had any effect on humoral immunity (Paulik et al. 1989).
Babiuk and Misra (1981 ) suggested that administration
of levamisole and attenuated infectious bovine rhino-
tracheitis vaccine in vivo did not elevate cellular or hu-
moral responses. Cabaj et al. (1995) reported similar
results in lambs injec ted with human erythrocytes and
ovalbumin 1 day after levamisole administration.
Most drugs’ actions on the immune system are
modulated by the interaction between the T cell
recruiting efficacy of the sulphur moiety and the cho-
linergic effects of the imidazole ring (Renoux 1980).
Levamisole appears to alter cyclic nucleotide phospho-
diesterases, decreasing cyclic guanosine monophosphate
(cGMP) degradation and increasing cyclic adenosine
monophosphate degradation (Boothe 2001). Stogause
and King (1995) suggested that, in the rat, levamisole
administration caused lower levels of corticosterone
than in controls and probably this reduction increased
immune function. Szeto et al. (2000) reported that lev-
amisole induces interleukin 18, a recently characterised
cytokine with potent activity in sti mulating interferon
gamma and shifting the immune balance toward a
type 1 response.
In conclusion our results indicated that, in normal
neonatal dairy calves, levamisole can promote non-spe-
cific immune responses, although further studies using
more precise methodology will be necessary.
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